A Predictable Change
A new technique can change a
plant's characteristics to make biofuels.
A core objective of Oak Ridge National Laboratory's BioEnergy Science Center is to find ways to wring more energy out of the sugars stored in plants. In addition to developing better enzymes, improved microbes and more effective catalysts, Gerald Tuskan's team of plant biologists is exploring ways to generate more energy from biomass by "persuading" plants to store more sugar and then developing new methods of extracting these sugars.
Most of the sugar found in biomass is stored in plant cell walls as cellulose and hemicellulose. The biggest roadblock to extracting sugar from these cell wall polymers has been the difficulty of using biochemical tools to break down the walls. Tuskan, a scientist in ORNL's BioSciences Division, is working with a dozen Oak Ridge scientists and university collaborators to study the genome of Populus, a group of trees commonly used in both biofuels research and biofuels production. The team is seeking to determine which of the plant's 42,000 genes play a role in the formation of the cell wall and which of those can be modified and regulated to create cell walls that are more easily broken down. "It's important to note that we're not talking about placing 'foreign' genes into Populus," Tuskan says. "We are modifying native genes that make it easier to break down the cell walls. We want to learn when these genes are turned on, how long they are on, and which plant tissues "
Currently Tuskan and his colleagues have narrowed to about 3580 the list of genes that might play a role in cell wall formation. They employ a variety of molecular techniques to study the genes and determine their respective functions. One of these techniques involves randomly inserting genetic "on" switches into the plant's genome to determine the effect. "Whatever gene the switch lands next to is turned on," Tuskan says. "We are inserting as many 'on' switches as possible to identify."
In about one quarter of the experiments, the technique produces a measurable change in the plant's physical characteristics. While some changes, such as coloration, are obvious to the naked eye, many are harder to discern. "The changes usually involve more subtle characteristics, like the level of sugar polymers in the plant's stem," Tuskan says. To detect these changes, Tuskan's team grows modified plants and then analyzes both their chemistry and their outward appearance. When a change is detected, researchers examine the plant's genome, locate the newly inserted "on" switches, and determine which gene or genes the switches have activated. The approach provides scientists with a good idea of which genes produced the changes. Once they know what a particular gene controls and whether it appears to play a role in the formation of the cell wall, researchers can make an informed decision about whether the gene should be turned "on" or "off" to improve the plant's usefulness for producing biofuels.
One of the challengs the team is addressing using this "on" switch technique involves disentangling the relationship between the molecules of cellulose and hemicellulose; polymers that make up the plant's cell walls; and lignin, a polymer that provides strength and stiffness to the plant's stalk. The standard biofuels pretreatment process involves relaxing the bonds between the cellulose and hemicellulose cell walls and lignin using a combination of high temperatures, dilute acid, and high pressure, so the lignin can be removed. Because the plant sugars are contained in the cellulose and hemicellulose, pretreatment removes the lignin but also has the undesired consequence of degrading the cellulose and hemicellulose, reducing the amount of stored sugar that can be captured.
A Predictable Change.
In search of a process that removes lignin from the biomass without sacrificing sugars, the team is looking for genes that can control the degree of polymerization of both lignin and cell walls. A more highly polymerized molecule has a more complex structure that tends to bind tightly to neighboring structures, making it more difficult to break down. This quality is known as recalcitrance. For the purposes of biofuels research, scientists would like to reduce the lignin's polymerization to facilitate its separation from the plant's cell walls. At the same time, researchers are seeking to increase the polymerization of cellulose and hemicellulose to enhance resistance to degradation during pretreatment. "Our basic goal is to reduce the recalcitrance of lignin and increase the proportion of intact cellulose," Tuskan says."All of our genetics work is being driven toward those two goals."
Selective breeding also plays a role in the team's efforts to develop an optimized strain of Populus. Drawing on the biochemical analysis of the 1100 genetically different trees in their collection, the researchers found that Populus's native lignin and sugar content vary greatly. "We have four individuals out of this collection that yield 87% of their theoretical maximum for sugar yield without any pretreatment at all," Tuskan says. "These are individuals we intend to either put in a breeding program or use for gene regulation research to determine the genetic basis for their favorable genetic characteristics."
Part of a process
A large part of what Tuskan and his team are trying to achieve is driven by the neighboring steps in the biofuels development process. Before they consider what kind of biomass would be suitable for producing biofuels, Tuskan and his colleagues have to examine issues of agronomy and sustainability. "The plants must be efficient in how they use water, nutrients, carbon and space. They need to be able to grow in a wide range of areas in a way that does not deplete the soil of key nutrients. We also want to be sure that farmers can grow these biomass crops in "
Similarly, much of the selective breeding and genetic fine-tuning Tuskan and his colleagues perform is aimed at making sure their final product has the sugar release, deconstruction and fermentation characteristics needed by the microbiologists who will be converting the plants to biofuels. "Our industrial collaborators are the ones most concerned with producing a sellable product," Tuskan explains. "Their short-term target is ethanol, but they are moving quickly toward higher-chain alcohols, like isobutanol or octanol. These fuels are preferable to ethanol because their energy per unit volume is similar to that of gasoline; they do not react with water; and they can be blended with gasoline in the current fuel distribution system."
Tuskan notes that when his group passes along a suitable feedstock to the bioconversion researchers, the process does not necessarily end. "We'll hear from them how well the biomass actually performs in the production process. We know how we expect the main polymers, cellulose, hemicellulose and lignin to perform, but there are other compounds in plants that can inhibit the biofuels production process. Before we know if we have a successful feedstock, we will have to see how it performs in an actual bioreactor."
"We hit the ground running"
Looking back at what has been accomplished by the BioEnergy Science Center in a relatively short time, Tuskan believes the center has made remarkable progress. "We really hit the ground running at BESC. Because we put an infrastructure together in a timely way, all the experiments are up and running. Usually with a project this size, there's a longer learning curve and a slow scale-up, but because of our team's experience and the quality of the participating institutions, we have exceeded our project milestones. Everything is in place for a period of genuine discovery."